Cyclic depsipeptides have numerous uses in pharmacology. As an example, the depsipeptides disclosed in WO2009/024527 are useful for treatment of various diseases. For example, the compound of formula II mentioned in WO2009/024527 is useful for the treatment and prevention of inflammatory and/or hyperpoliferative and pruritic skin diseases such as atopic dermatitis, psoriasis, pustular psoriasis, rosacea, keloids, hypertrophic scars, acne, Netherton's syndrome or other pruritic dermatoses such as prurigo nodularis, unspecified itch of the elderly as well as other diseases with epithelial barrier dysfunction such as aged skin.
Nostopeptin BN920, formerly isolated from the cyanobacterium Nostoc, was isolated also from Microcystis. Nostopeptin BN920 inhibited chymotrypsin with an IC50 value of 31 nM (see J. Nat. Prod. 68(9), 1324-7 (2005)).
These compounds can be produced by fermentation (using chondromyces croactus, myxobacteria) along with other depsipeptides comprising the so-called ahp-substructure (ahp: 3-amino-6-hydroxy-piperidin-2-one) and the corresponding dehydro-ahp substructure (dehydro-ahp: 3-amino-3,4-dihydro-1H-pyridin-2-one), also called “dehydrate” herein, respectively. Therefore, the yield of fermentation with regard to any single of these compounds is rather low.
Hitherto, the synthesis of such compounds was based on solution chemistry approaches or in copending PCT application No. PCT/IB2012/051977 by a combination of solid phase and solution peptide chemistry.
A critical step is the formation of the ahp-substructure. This, according to published prior art, is mainly formed by oxidation of the open chain precursor amino acid 2-amino-5-hydroxy-pentanoic acid in the closed macrolactone ring by oxidative treatment via a labile aldehyde intermediate (see e.g. Yokohama et al., Tetrahedron 61 (2005), pp. 1459-80, compound 23; Yokohama et al., Pept. Sci. 38 (2002). Pp. 33-36; and Yokohama et al., Tetrahedron Lett. 42 (2001), 5903-8).
The aldehyde is too instable to be isolated. Therefore its direct use and synthesis are not recommended.
Aldehyde derivatives, such as acetals, are also known to be instable, in particular in the case where acetal and (especially free) carboxylic acid functions are present simultaneously or under (even only slightly) acidic conditions.
There is a need to find higher yielding processes and processes that are easier in handling for the manufacture of macrolactone ring systems comprising ahp moieties.
It has now been found that it is possible to replace the precursor with the 2-amino-5-hydroxypentanoic acid building block and use its 5-oxo-analogue in acetal form instead.
The present invention thus relates to processes or methods that allow obtaining such cyclic depsipeptides with increased yield and/or in good purity and with a lower number of steps.
In view of the many risks, such as racemization, tautomerization and the like, in the synthesis of a complex molecule with many possible isomers, it has been possible to find a manufacturing process, preferably comprising a mixture of solid phase peptide synthesis and reactions in solution, that allows to produce cyclic depsipeptides of formula I in good yield and/or with the required stereoisomerical purity, especially both, and that avoids the steps of oxidation of a hydroxyl group in the precursor molecule. It is possible to reduce the amount of by-products, and even to improve yield, by converting such by-products, especially the dehydro-ahp substructure and/or an analogue of the desired ahp-comprising products with a five-ring instead of the ahp, into the desired final products. No synthesis has so far come to our attention making use of solid phase peptide synthesis in this field. In addition, the elimination of the oxidation step allows using N-Me-Tyosine or analogues instead of the protected variants such as t-butyl-ethers or analogues thereof which are expensive and difficult to prepare.
(i/a) In a first embodiment, the invention relates to a method or process for the preparation of a cyclic depsipeptide compound of the formula I,
especially of the formula IA
whereinA1 is a bivalent moiety of an amino acid with a terminal carboxy or carbamoyl group, especially asparagine or glutamine, and is bound at its right hand side in formula I via a carbonyl (preferably the carbonyl of an α-carboxyl group thereof) to the rest of the molecule; or is C1-8-alkanoyl or phosphorylated hydroxy-C1-8-alkanoyl;X is bound via an N of A1 and is acyl, or is absent if A1 is C1-8-alkanoyl or phosphorylated hydroxy-C1-8-alkanoyl;R2 is C1-8-alkyl, especially methyl;R3 is the side chain of an amino acid, especially of leucine, isoleucine or valine;R5 is the side chain of an amino acid, preferably of phenylalanine, leucine, isoleucine or valine;R6 is the side chain of a hydroxy amino acid, especially of tyrosine;R7 is the side chain of an amino acid, preferably of the amino acid leucine, isoleucine or valine; andY is hydrogen or C1-8-alkyl;or a salt thereof,said method comprisingdeprotecting a compound of the formula II
especially of the formula IIA
wherein the aldehyde protecting group(s) Rk and Rl are independently of each other unsubstituted or substituted alkyl or together with the two binding O atoms and the carbon atom to which the two O atoms are bound form a ring that is unsubstituted or substituted (Rk and Rl then preferably forming an unsubstituted or substituted alkylene bridge, especially unsubstituted or substituted ethylen, such as —CH2—CH2— or —CH2—CH2—CH2—), Y is as defined for a compound of the formula I and X*, A1*, R2*, R3*, R5*, R6*, and R7* correspond to X, A1, R2, R3, R5, R6, and R7 in formula I, respectively, but with the proviso that reactive functional groups on these moieties (such as amino, imino, hydroxy, carboxy, sulfhydryl, amidino, guanidino, O-phosphono (—O—P(═O)(OH)2) are present in protected form at least if they could participate in undesired side reactions, to result in a compound of the formula I, especially IA;and, if desired, converting a free compound of the formula I, or especially IA, into a salt, a salt of a compound of the formula I into a different salt of a compound of the formula I, or especially IA, or into the free compound of the formula I, or especially IA, and/or converting a dehydrate analogue and/or five ring analogue of a compound of the formula I, or especially IA, into the corresponding compound of the formula I, or especially IA.(ii/a) A further embodiment of the invention refers to the method or process described above, in addition comprising manufacturing the compound of the formula II or especially IIA by a combination of Solid Phase Peptide Synthesis (especially for synthesis of the precursor of formula III or III* or especially IIIA or IIIA* given below, and Solution Phase synthesis (especially from the compounds just mentioned to the final product) from the corresponding starting amino acids and side chain precursors.(i/b) Yet a further embodiment of the invention relates to a method or process as described above, further comprising, for the synthesis of a compound of the formula II above, cyclization under lactamization (macrolactamization) of a linear precursor peptide of the compound of the formula II or especially of the formula IIA, carrying an N-terminal amino group and a C-terminal carboxy group, under reaction conditions that allow for the formation of an amide bond from said amino and said carboxy group, preferably using Solution Phase chemistry.(ii/b) A further embodiment of the invention relates to the method or process according to the preceding paragraph (i/b), where the linear precursor peptide is of the formula III,
especially IIIA,
wherein Rk, Rl, X*, A1*, R2*, R3*, R5*, R6* and R7* are as defined for a compound of the formula II above, which can be obtained directly from solid phase peptide synthesis (e.g. as described under step (iii/b) or by deprotection from the corresponding compound of the formula III*,
especially IIIA*,
wherein Rk, Rl, X*, A1*, R2*, R3*, R5*, R6* and R7* are as defined for a compound of the formula II above and wherein each of the Prot** moieties is a protecting group that can preferably must) be removed under conditions different from those of the cleavage under (iii/b), especially each is an arylalkyl amino protecting group, as defined for a compound of the formula IV, by deprotecting the protected amino group.(iii/b) Another embodiment refers to the method or process according to the preceding paragraph (ii/b), further comprising, for the synthesis of the compound of the formula III or especially IIIA, or of a compound of the formula III*, especially IIIA*, cleaving a compound of the formula IV,
especially IVA,
wherein Rk, Rl, X*, A1*, R2*, R3*, R6*, R6* and R7* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, n is a natural number not including 0 andZ is a protected amino group either of the formula NHProt* wherein Prot* is a protecting group that is removed before or during the cleaving reaction or further subsequently to it [?] to yield a compound of the formula III, especially IIIA; or Z is a protected amino group of the formula N(Prot**)2 wherein each Prot** is an amino protecting group that can (in particular can only) be removed under conditions different from those of the cleaving reaction, especially each is an arylalkyl amino protecting group, to yield the compound of the formula III*, especially IIIA*.(iv/b) A further embodiment of the invention relates to the method or process according to the preceding paragraph (iii/b), further comprising, for the synthesis of the compound of the formula IV, especially IVA, coupling an amino acid of the formula V,
especially VA,
wherein Rk and Rl are as defined for a compound of the formula II above and Z is a protected amino group either of the formula NHProt* wherein Prot* is a protecting group that can be (and is) removed before or during the cleaving reaction under (iii/b) or further subsequently; or Z is a protected amino group of the formula N(Prot**)2 wherein each Prot** is an amino protecting group that can be removed under conditions different to those of the cleaving reaction under (iii/b), especially each is an arylalkyl amino protecting group; or an activated derivative of said amino acid of the formula V or VA, with a compound of the formula VI,
especially VIA,
wherein X*, A1*, R2*, R3*, R5*, R6* and R7* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number not including 0.(v/b) Yet a further embodiment of the invention relates to the method or process according to the preceding paragraph (iv/b), further comprising, for the synthesis of the compound of the formula VI, especially VIA, coupling an amino acid of the formula VII
especially VIIA,
wherein R5* is as defined for a compound of the formula II above and Prot*** is an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the coupling product remaining on the resin, or a reactive derivative of said amino acid, with a compound of the formula VIII,
especially VIIIA
wherein X*, A1*, R2*, R3*, R6* and R7* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number not including 0, and removing the protecting group Prot***.(vi/b) In yet a further embodiment, the invention relates to the method or process according to the preceding paragraph (v/b), further comprising, for the synthesis of the compound of the formula VIII, especially VIIIA, coupling an amino acid of the formula IX,
especially IXA
in which R6* and Y are as defined for a compound of the formula II above and Prot*** is an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the coupling product remaining on the resin, or a reactive derivative of said amino acid, with a compound of the formula X,
especially XA,
wherein X*, A1*, R2*, R3* and R7* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number not including 0, and removing the protecting group Prot***.(vii/b) Another embodiment of the invention relates to the method or process according to the preceding paragraph (vi/b), further comprising, for the synthesis of a compound of the formula X, especially XA, reacting an amino acid of the formula XI,
especially XIA,
wherein Prot*** is an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin, and R7* is as defined for a compound of the formula II above, or a reactive derivative of said amino acid,with the hydroxyl group of a compound of the formula XII,
especially XIIA,
wherein X*, A1*, R2* and R3* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number not including 0; and removing the protecting group Prot***.(viii/b) In a further embodiment, the invention relates to the method or process according to the preceding paragraph (vii/b), further comprising, for the synthesis of a compound of the formula XII, especially XIIA, coupling a resin bound dipeptide symbolized by the formula XIII,
especially XIIIA
in which Prot**** is a protecting group that can be cleaved off selectively without affecting other protecting groups present in a compound of the formula II as defined above and with the product remaining on the resin, R2* and R3* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number not including 0, after removal of the protecting group Prot**** via the thus obtainable free amino group, with an amino acid of the formula XIV,
in particular of the formula XIV*,
more particularly of the formula XIV**,
wherein X** is an amino protecting group or is X*, and wherein X* and A1* are as defined for a compound of the formula II above, or a reactive derivative of said acid;and, if X** is an amino protecting group, removing said amino protecting group X** to yield the derivative of formula II wherein, instead of X*, H is present and coupling the resulting amino group with an acyl group X* using the corresponding acid X*—OH wherein X* is as defined for a compound of the formula II above, or a reactive derivative of said acid.(ix/b) A yet further embodiment of the invention relates to the method or process according to the preceding paragraph (viii/b), further comprising, for the synthesis of a compound of the formula XIII, especially XIIIA, coupling a resin bound amino acid symbolized by the formula XV,
especially XVA,
wherein R3* is as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number not including 0,with an amino acid of the formula XVI,
especially XVIA,
wherein Prot**** is a protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin, and R2* is as defined for a compound of the formula II above, or a reactive derivative of said amino acid.(x/b) A further embodiment of the invention relates to the method or process according to the preceding paragraph (ix/b), further comprising, for obtaining the resin bound amino acid of the formula XV, especially XVA, coupling an amino acid of the formula XVII,
especially XVIIA,
wherein R3* is as defined for a compound of the formula II above or below and Prot*** is an amino protecting group can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin; or a reactive derivative of said amino acid of the formula IX, to a cleavable linker L which is bound to a solid resin RES, and removing the protecting group Prot***.(i/c) Another embodiment of the invention relates to the method or process according to any one of the preceding paragraphs (i/a) to (x/b) where the symbols A1, R2, R3, R5, R6, R7, X and Y or the corresponding unprotected or protected moieties R2*, R3*, R5*, R6*, R7*, X* and Y are selected so that, in the resulting compound of the formula I, or a salt thereof,A1 is the bivalent radical of L-glutamine bound via the carbonyl of its α-carboxy group to the amino group at the right of A1 in formula I and via its α-amino group to X, or is 2S-(2-hydroxy-3-phosphonooxy)-propionyl;R2 is methyl;R3 is isopropyl, isobutyl (2-methyl-n-propyl wherever used), especially isobutyl;R5 is sec-butyl or benzyl, especially sec-butyl;R6 is 4-hydroxybenzyl;R7 is isopropyl or sec-butyl (1-methyl-n-propyl wherever used), especially sec-butyl;X is acetyl or isobutyryl, or is absent if A1 is 2S-(2-hydroxy-3-phosphonooxy)-propionyl andY is methyl.(i/d) In another particular embodiment, the invention relates to a method or process for converting a dehydrate obtained from a compound of the formula II, especially IIa, of a compound of the formula I given obtained from a compound of the formula II above or in particular with the substituents as defined in the preceding paragraph (i/c) into the corresponding compound of the formula I, where the dehydrate has the formula XVIII,
especially XVII IA,
in which Y, X, A1, R2, R3, R5, R6 and R7 are as defined for a compound of the formula I above;or especially a method or process for shifting the equilibrium of a mixture of a compound of the formula I and its corresponding dehydrate, and/or its corresponding hemiaminal analogue with a five-ring instead of the ahp structure in formula I which may also be formed as byproduct and has the formula XIX,
especially the formula XIXA,
in which Y, X, A1, R2, R3, R5, R6 and R7 are as defined for a compound of the formula I above, respectively;in favor of the compound of the formula I,said method or process comprising using an aqueous acid as reactive solvent to drive the reaction. This method is especially used in addition to the other processes or methods described above and below to increase the yield or to re-convert a compound of the formula V, especially VA, and/or the analogue with a five-membered ring instead of the ahp structure in formula I, into the corresponding compound of the formula I.
The method described for the conversion of the dehydrate and/or the five ring analogue (always regarding the desired ahp ring) into the desired compound of the formula I or especially IA, e.g. of Compound A-dehydrate from Example 3B into Compound A, enables a straight-forward synthesis of this class of compounds. Up to now, an acidic treatment as final step had to be circumvented in order to avoid the dehydration of the product.
(i/e) A further embodiment of the invention relates to the method according to the preceding paragraph (i/d), wherein the acid is a carboxylic acid, especially a halo substituted C1-8alkanoic acid, more especially trifluoroacetic acid or trichloroacetic acid.(i/f) The invention, in yet a further embodiment, relates to a compound of the formula XX,
especially of the formula XXA,
wherein Rk and Rl are as define d for a compound of the formula II above, Y is as defined for a compound of the formula I in the first instance above or in particular as defined above under (i/c) and X*, A1*, R2*, R3*, R5*, R6*, and R7* correspond to X, A1, R2, R3, R5, R6, and R7 in formula I as defined above or below or in paragraph (ia) or especially under (i/c) given above, respectively, however with the proviso that reactive functional groups on these moieties are preferably present in protected form.(i/g) In a further embodiment, the invention relates to a novel compound selected from the group consisting of compounds of the formula II, III, III*, IV and V, and especially of the formula IIA, IIIA, IIIA*, IV and VA yet more especially to the group consisting of the following (especially enantiomerically enriched or pure) compounds: Compound 2*, Compound 3*, compound 5*, preferably Compound 2A*, Compound 3A*, Compound 4A*, Compound 5A*, especially in the form given in the examples: Compound 2, Compound 3, Compound 5, Compound 6, Compound 7, Compound 8, Compound A, Compound 9 and Compound 10 and enantiomerically enriched or especially pure Compound 4, as well as compounds 12, 14, 15, 16 and 17. Also preferred are the compounds of the formulae 12*, 12A*, 14*, 14A*, 15*, 15A*, 16*, 16A*, 17* or 17A* given below, in which Rl and Rk independently of each other are 1-aralkyl, such as 1-(C6-C12aryl)-C1-C7alkyl, more especially benzyl, as well as the corresponding compounds wherein Rl and Rk together form an unsubstituted or substituted alkylene, especially—CH2—CH2— or —CH2—CH2—CH2—.(i/h) In yet a further embodiment, the invention relates to a method or a process for the synthesis of a compound of the formula V, especially of the formula VA mentioned above, according to either                (a) (especially) in the case of the synthesis of a compound of the formula V wherein Rk and Rl together form an unsubstituted or substituted lower alkylene bridge, especially —CH2—CH2— or —CH2—CH2—CH2—, the following reaction scheme, alternatively via the route (i) 1*→2*→3*, (ii) 1*→2*→4*→5*, or (iii) 1*→2*→3*→5*:        
(wherein Rk, Rl have the meanings just indicated and Z has the meanings mentioned above for a compound of the formula V, especially in the compounds 1*, 2* and 3* being N(Prot**)2 as defined for a compound of the formula V, especially 1-(C6-C12-aryl)-C1-C6-alkyl, especially benzyl, and in the compound 5* being NHProt* as defined for a compound of the formula V, Prot* especially being an acyl protecting group, e.g. fluoren-9-yl-methoxycabonyl, and the compounds of formula 3* and 5* each correspond to a compound of the formula V;or especially, to obtain a compound of the formula VA, the following scheme, alternatively via the route (i) 1A*→2A*→3A*, (ii) 1A*→2A*→4A*→5A*, or (iii) 1A*→2A*→3A*→5A*:
(wherein Rk, Rl have the meanings just indicated and Z and Prot* have the meanings mentioned above for a compound of the formula V, especially Z in the compounds 1A*, 2A* and 3A* being 1-(C6-C12-aryl)-C1-C6-alkylamino, especially benzylamino, and Prot* in the compound 5A* being an acyl protecting group, e.g. fluoren-9-yl-methoxycabonyl, where the compounds of formula 3A* and 5A* each correspond to a compound of the formula VA;where the reaction of 1* or 1A* to 2* or 2A* is an acetal formation reaction with an unsubstituted or substituted lower alkylendiol, especially ethylene glycol, e.g. in an appropriate solvent, such as dichloromethane, in the presence of an acid, such as toluene sulfonic acid, in the presence of e.g. molecular sieve; the reaction of 2* or 2A* to 3A or 3A* by hydrolysis in the presence of a base, such as an alkali metal hydroxide, e.g. LiOH, in an ether, e.g. dioxane, and water; or the alternative reaction from 2*, especially 2A* to 4*, especially 4A*, under deprotection of the carboxy and the amino group is made by e.g. catalytic hydrogenation, e.g. hydrogenation with a noble metal catalyst, e.g. Pd or Pt, e.g. on a carrier such as aluminium oxide or carbon, an appropriate solvent, e.g. an alcohol, such as methanol, ethanol or isopropanol, followed by reintroduction of an amino protecting group Prot**, especially an acyl protecting group, such as fluoren-9-yl-methoxycarbonyl, e.g. under acylation conditions or in the presence of a coupling agent as mentioned below for amino acid or acid couplings to amino groups, especially using the (e.g.Fmoc-) HOSU-ester, a tertiary base, e.g. triethylamine, and an appropriate solvent, e.g. water and/or acetonitrile; where alternatively the compound of formula 4*, especially 4A*, may be obtained from a compound of the formula 3*, especially 3A*, by catalytic hydrogenation as just mentioned;and where the compound 1* or 1A* may be obtained by or in analogy to the method mentioned in Rodriguez and Taddei, Synthesis 2005, 3, pp. 493-495);or                (b) (especially in the case of the synthesis of a compound of the formula V, especially VA mentioned above, wherein each of Rk and Rl is an unsubstituted or substituted alkyl moiety, especially 1-aralkyl, such as 1-(C6-C12aryl)-C1-C7alkyl, more especially benzyl) according to the following reaction scheme:        
(wherein Rk, Rl have the meanings just indicated and Prot* has the meanings mentioned above for a compound of the formula V, especially in the compounds 17* being an acyl protecting group, e.g. fluoren-9-yl-methoxycabonyl, and the compounds of formula 17* correspond to a compound of the formula V;or especially, to obtain a compound of the formula VA, the following scheme:
(wherein Rk, Rl have the meanings just indicated and Prot* has the meanings mentioned above for a compound of the formula V, especially in the compounds 17A* being an acyl protecting group, e.g. fluoren-9-yl-methoxycabonyl, and the compounds of formula 17A* correspond to a compound of the formula VA;where preferably the reaction of 11 with 12 or 12A* takes place in an appropriate solvent, e.g. methylene chloride, with a brominating agent, e.g. trimethylbromosilane, followed by the addition of compounds of the formula Rk-OH and Rl-OH (which are preferably identical) and a mixture or tertiary base, e.g. pyridine and N,N-dimethylaminopyridine, and an acid anhydride, e.g. acetic anhydride; reaction of the compound 12* or 12* with a compound of the formula 13* or 13A* in an appropriate solvent, e.g. an acid amide, such as dimethylformamide, a strong base, e.g. potassium tert-butoxide; hydrolysis of the resulting compound of the formula 14*, especially 14A*, with an alkali metal hydroxide, e.g. potassium hydroxide, in an appropriate solvent, e.g. an alcohol, such as ethanol, and subsequent warming up for decarboxylation to give a compound of the formula 15*, especially 15A*, removal of the acetyl group in an appropriate buffer, e.g. in the range of pH 7 to 10, e.g. in aqueous citrate buffer titrated with an alkali metal hydroxide, such as sodium hydroxide, with an acylase, if required in the presence of cofactors such as cobalt chloride, to give a compound of the formula 16*, especially 16A*; and introduction of a protecting group Prot**, e.g. acyl, e.g. 9-fluorenylmethoxycarbonyl, with an appropriate reagent, e.g. Fmoc-OSU, in an appropriate solvent, e.g. water and/or acetonitrile.
The following definitions (or also definitions already included above) can replace more general terms used in invention embodiments above and below in order to define further embodiments of the invention, with either one, two or more or all general terms being replaceable by the more specific terms in order to define such invention embodiments:
In all reactions, protecting gas may be used, such as nitrogen or Argon, where appropriate or necessary, and the temperatures are as known to the person skilled in the art, e.g. in the range from −25° C. to the reflux temperature of the respective reaction mixture, e.g. from −20 to plus 90° C.
If Rk and Rl are each independently of each other unsubstituted or substituted alkyl, this refers especially to C1-C7-alkyl or especially 1-aralkyl, such as 1-(C6-C12aryl)-C1-C7alkyl, more especially benzyl.
If Rk and Rl together with the two binding O atoms and the carbon atom to which the two O atoms are bound form a ring that is unsubstituted or substituted, Rk and Rl then preferably form an unsubstituted or substituted alkylene bridge, especially unsubstituted or substituted ethylen, such as —CH2—CH2—), where the substituent(s) may preferably be selected from C1-C7-alkyl, especially two such substituents, such as methyl, ethyl, n-propyl or isopropyl.
A bivalent moiety of an amino acid with a terminal carboxy or carbamoyl group is preferably an alpha-carbamoyl or carboxyl-C1-8-substituted amino acid, especially the bivalent moiety of asparagine or glutamine, and is bound at its right hand side in formula I via a carbonyl (preferably the carbonyl of its α-carboxyl group) to the rest of the molecule.
C1-8-alkanoyl or phosphorylated hydroxy-C1-8-alkanoyl (C1-8-alkanoyl carrying both a hydroxyl and a phosphono (—O—P(═O)(OH)2) group) A1 is e.g. 2,3-dihydroxy-propanoyl (preferably in S-form) or 2-hydroxy-3-phosphono-propanoyl (preferably in S-form).
R2 and R2* are C1-8-alkyl, especially methyl wherever mentioned.
R3 is the side chain of an amino acid, especially of a natural amino acid. Preferably, it is C1-8alkyl which may be branched or linear. Most especially, C1-8alkyl is n-(2-methyl)propyl (isobutyl), n-(1-methylpropyl (sec-butyl) or methyl, that is, the amino acid carrying the moiety is leucine, isoleucine or valine.
R3* is the corresponding side chain in protected form if a functional group is present that has to be hindered to participate in a reaction. Preferably, it is C1-8alkyl which may be branched or linear, especially as defined in the preceding paragraph.
A “side chain of an amino acid” may be selected from any moiety, e.g. a mono- or polycyclic, linear, saturated, unsaturated (e.g. with conjugated double bonds) or partially saturated organic moiety, e.g. with up to 20 carbon atoms and 0 to 5 heteroatoms in the basis structure independently selected from N, O and S replacing the corresponding number of carbon atoms, and may be substituted by up to three moieties selected from amino, imino, hydroxy, carboxy, carbamoyl, sulfhydryl, amidino, guanidino, O-phosphono(—O—P(═O)(OH)2). Preferably, the side chains are selected from those of the 20 standard alpha-amino acids arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, valine and further proline (then with internal cyclization including the alpha-amino group).
For the amino acids, either their names or the customary three letter codes are used in the present disclosure, in accordance with the following table:
Amino acidThree letter codeAlanineAlaArginineArgAsparagineAsnAspartic acidAspAsparagine or aspartic acidAsxCysteineCysGlutamic acidGluGlutamineGlnGlutamine or glutamic acidGlxGlycineGlyHistidineHisisoleucineIleLeucineLeuLysineLysMethionineMetPhenylalaninePheProlineProSerineSerThreonineThrTryptophanTryTyrosineTyrValineVal
R5 is the side chain of an amino acid, preferably a standard amino acid. Preferably, it is C1-8alkyl which may be branched or linear and which is unsubstituted or substituted by phenyl. Most especially it is benzyl, n-(2-methyl)propyl, isobutyl or methyl, that is, the amino acid carrying the moiety is phenylalanine, leucine, isoleucine or valine.
R6 is the side chain of a hydroxy amino acid, especially of tyrosine.
R7 is the side chain of an amino acid, especially of a natural amino acid. Preferably, it is C1-8alkyl which may be branched or linear. Most especially it is n-(2-methyl)propyl(isobutyl), n-(1-methyl)propyl (sec-butyl) or methyl, that is, the amino acid carrying the moiety is leucine, isoleucine or valine.
C1-8-alkyl can be linear or branched one or more times; for example, it can be n-(2-methyl)propyl, n-(1-methyl)propyl or methyl.
All of the compounds can, where salt-forming groups such as basic groups, e.g. amino or imino, or acidic groups, e.g. carboxyl or phenolic hydroxyl, are present, be used in free form or as salts or as mixtures of salts and free forms. Thus where ever a compound is mentioned, this includes all these variants. For example, basic groups may form salts with acids, such as hydrohalic acids, e.g. HCl, sulfuric acid or organic acids, such as acetic acid or trifluoroacetic acid, while acidic groups may form salts with positive ions, e.g. ammonium, alkylammonium, triethylamine, N-methylmorpholine, dimethylaminopyridine, alkali or alkaline-earth metal salt cations, e.g. Ca, Mg, Na, K or Li cations, or the like, or zwitterionic salts or inner salts of the compounds may be present.
“Or the like” or “and the like”, wherever used in this disclosure, refers to the fact that other alternatives to those mentioned preceding such expression are known to the person skilled in the art and may be added to those expressions specifically mentioned; in other embodiments, “or the like” and “and the like” may be deleted in one or more or all invention embodiments.
Acetal protecting groups are highly sensitive to acidic conditions, especially in the presence of water. Cleavage of the acetal protecting group during the solid phase peptide synthesis or during cleavage from solid support would generate the free aldehyde function, which could react with the free amino group and undergo other side reactions. Therefore, it is important to keep the acetal protecting group until the cyclization of the oligopeptide is performed to obtain the macrocyclic compound II or IIA.
The protecting groups Prot**, Prot***, Prot**** and any further protecting groups present on the moieties A*, R2*, R3*, R5*, R6*, R7*, X*, where ever mentioned throughout the present description and claims, are selected so that they allow for orthogonal protection.
Orthogonal protection is a strategy allowing the deprotection of multiple protective groups one (or more but not all) at the time where desired each with a dedicated set of reaction conditions without affecting the other protecting group(s) or bonds to resins, e.g. via linkers on solid synthesis resins. In other terms: The strategy uses different classes of protecting groups that are removed by different chemical mechanisms, also using appropriate linkers in the case of solid phase peptide synthesis (where the linker-resin bond might together be considered as a carboxy protecting group).
Preferably, the protecting groups are selected as follows:
Prot* (a protecting group that can be removed (=is appropriate for removal) during the cleavage of IV, especially IVA, during the reaction under (iii/b) or subsequently but, on the other hand, can be removed on the resin without cleaving other bonds (no cleavage of an amino acid or peptide bound via the carbonyl of its (especially α-carboxyl group to the binding via a linker L mentioned below; also without cleaving off other protecting groups present), especially a protecting group removable without cleavage of an ester (instead of an amide) bond in a depsipeptide or depsipeptide precursor and under conditions other than those for other protecting groups present, while preserving the binding via the linker to a resin RES where present; it is preferably removable by a mild base, e.g. piperidine, morpholine, dicyclohexylamine, p-dimethylamino-pyridine, diisopropylamine, piperazine, tris-(2-aminoethyl)amine in an appropriate solvent, e.g. N,N-dimethylformamide, methylene chloride; Prot** is, e.g., selected from the group consisting of fluoren-9-ylmethoxycarbonyl (Fmoc); 2-(2′ or 4′-pyridyl)ethoxycarbonyl and 2,2-bis(4′ nitro-phenyl)ethoxycarbonyl.
Prot** is a protecting group that can be removed (especially from a compound of the formula III* or especially IIIA*) under conditions that are different to those of the cleaving reaction under (iii/b) especially arylalkyl, especially 1-(C6-C12aryl)-C1-C4alkyl, more especially benzyl, which can be removed e.g. by catalytic hydrogenation, e.g. with hydrogen in the presence of a noble metal catalyst, such as Pd or Pt which may be on a carrier, such as aluminium oxide or especially carbon.
Prot*** (an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin) is selected from those mentioned for Prot*, e.g. fluoren-9-ylmethoxycarbonyl (Fmoc), each of which can be removed e.g. as mentioned above or below.
Prot**** is a protecting group that can be cleaved off selectively without affecting other protecting groups present, especially as defined for Prot***.
The preferred orthogonal synthesis method in this case makes use of the Fmoc-protecting group strategy known in general for peptide synthesis using solid phase and liquid phase peptide synthesis.
The aldehyde protecting group(s) Rk and Rl (which together with the binding O atoms and the carbon binding them form a protected aldehyde group (an acetal) can be removed in the presence of water by acid catalysis, especially an alpha-halo substituted alkanoic acid, such as trifluoroacetic acid or trichloroacetic acid.
Other protecting groups present as well as the binding linker to a resin RES where present are preferably not removable under conditions under which Prot*, Prot**, Prot*** and Prot**** can be removed, e.g. in A*, carbamoyl can be N-protected e.g. with trityl (triphenylmethyl) (cleavage e.g. with trifluoro acetic acid (TFA); (e.g. in R6*) a tyrosine hydroxy can be Boc (tert-butoxycarbonyl) protected, or protected by tert-butyldimethyl-silyl, methoxymethyl or arylacetate (cleavage e.g. with TFA) and more preferably under conditions under which the bond to the linker to the Resin RES is preferably not cleaved or (where simultaneous deprotection and cleavage from the resin to the bond is desired) also cleaved (e.g. cleavage with acid, such as TFA).
Appropriate protecting groups are known in the art, as well methods for their introduction and removal. For example, the protecting groups, their introduction and removal methods may be selected from those described in standard textbooks such as “Protective Groups in Organic Synthesis”, 3rd ed., T. W. Green and P. G. M. Wuts (Eds.). J. Wiley & Sons, Inc., New York etc. 1999.
The protecting groups Prot*, Prot**, Prot***, Prot**** and other protecting groups are thus not limited to those mentioned above—rather they should fulfill conditions that make them appropriate for orthogonal protection, e.g. as described above or below.
It is recommended to avoid too basic conditions (though the bases described for Fmoc cleavage, such as piperidine, are usually allowable) to avoid cleavage of the depsipeptide (ester) bond.
An appropriate solvent or solvent mixture useful during the deprotection steps may, e.g., be selected from customary solvents, e.g. an N,N dialkylformamide, such as dimethylformamide, a halogenated hydrocarbon, e.g. dichloromethane, alkanols, such as ethanol, propanol or isopropanol, nitriles, e.g. acetonitrile, alkanoic acid amides, such as dimethylformamide or diethylformamide, or further an aromatic hydrocarbon, e.g. toluene, or mixtures of two or more, also water may be present. The temperatures may be ambient temperature or lower or higher, e.g. in the range from −20° C. to 50° C.
Among the possible solid support for Solid Phase Peptide Synthesis (SPPS), the following may be mentioned:                Gel-type supports without or with spacer: These are highly solvated polymers with an equal distribution of functional groups. This type of support is the most common, and includes:        Polystyrene: Styrene cross-linked with e.g. 1-2% divinylbenzene; Polyacrylamide or polymethacrylamide: as hydrophilic alternative to polystyrene; Polyethylene glycol (PEG): PEG-Polystyrene (PEG-PS) is more stable than polystyrene and spaces the site of synthesis from the polymer backbone; PEG-based supports: Composed of a PEG-polypropylene glycol network or PEG with polyamide or polystyrene (these already include a spacer, PEG);        Surface-type supports: Materials developed for surface functionalization, including controlled pore glass, cellulose fibers, and highly cross-linked polystyrene.        Composites: Gel-type polymers supported by rigid matrices.        
Usually these gels carry reactive groups to which a linker L as mentioned for various precursors above and below can be bound. For example, such groups include aminomethyl groups, polyethyleneglycol groups with a terminal hydroxy, and the like.
Any such support can be used in the embodiments of the present invention.
Gel type supports are used in another special embodiment of the invention, Among these, polystyrene (divinylbenzene crosslinked); polyacrylamide and polymethacrylamide resins are especially preferred.
Among the possible linkers, all commonly known and appropriate may be used.
Examples in possible embodiments of the invention are the 2-methoxy-4-benzyloxy-benzyl alcohol linker (a Sasrin-Linker, Sasrin stands for superacid sensitive resin, binds the amino acids or peptides via alcoholic OH); the trityl linker family (e.g., Trityl, 2Cl-Trityl, which bind the amino acids or peptides via OH); the 4-(2,4-dimethoxyphenylhydroxy-methyl)phenoxymethyl-Linker (Rink-Acid-Linker, binds the amino acids or peptides via OH); or tris(alkoxy)benzyl ester linkers (HAL-Linker, binds the amino acids or peptides via OH).
The introduction of linker groups and their coupling with amino acids can be conducted essentially as described or in analogy to the Examples. For example, in the case of trityl ester formation, the resin (e.g. divinylbenzene cross-linked aminomethylpolystyrene resin) may be suspended in an appropriate solvent, such as a dialkyl acid amide, e.g. dimethylformamide, and/or an alcohol, such as ethanol, propanol or isopropanol, and reacted with a hydroxyaryl-acid linker, e.g. 4-(diphenylhydroxymethyl)-benzoic acid, in the presence of a coupling agent, e.g. mentioned below for the coupling of acids, e.g. 1-hydroxybenzotrialzoe and dicyclohexycicarbodiimide; or, for the manufacture on chloro-(2′ chloro)triytl-polystyrene resin, the resin is suspended in an appropriate solvent, e.g. dichloromethane, addition of a chlorinating agent, e.g. acetyl chloride, and then reaction with the carboxyl group of an amino acid (this term always including unprotected or protected amino acids), e.g. in the presence of a base, e.g. a tertiary amino base, such as N-methyl-morpholine.
The cleavage of completed (protected or unprotected) peptides, e.g. to achieve the linear precursor peptide of formula III, especially IIIA, or III*, especially III*A, can then be conducted under mild acidic conditions, e.g. in the presence of an organic alkanoic acid, such as acetic acid, in an appropriate solvent, e.g. in dichloromethane or trifluoroethanol.
The cleavage conditions from the solid support must be selected such that the other protecting groups present in the molecule such as the trityl-, t-butyl- and in particular the acetal-protecting groups are not cleaved. Acetal protecting groups are highly sensitive to acidic conditions, especially in the presence of water. Cleavage of the acetal protecting group during the solid phase peptide synthesis or during cleavage from solid support would generate the free aldehyde function, which could react with the free amino group and undergo other side reactions.
Where reactive derivatives of acids, especially amino acids, or peptides, e.g. dipeptides, are mentioned, they may be formed in situ or may be used as such.
Reactive (or active) derivatives used as such include the halogenides, e.g. chlorides, or nitrophenyl esters, e.g. the 2,4-dinitrophenyl esters, or acid anhydrides (symmetric or e.g. with acetic acid) of the carboxy groups of the acids to be reacted.
For in situ formation, customary coupling agents may be applied. Such reagents are known to the person skilled in the art and can be deduced conveniently from many sources, e.g. Aldrich ChemFiles—Peptide Synthesis (Aldrich Chemical Co., Inc., Sigma-Aldrich Corporation, Milwaukee, Wis., USA) Vol. 7 No. 2, 2007 (see hftp://www.sigmaaldrich.com/etc/medialib/docs/Aldrich/Brochure/al_chemfile_v7_n2. Par. 0001.File.tmp/al_chemfile_v7_n2.pdf). Among the possible coupling agents for amide and ester bond synthesis the following may be mentioned:
Triazoles, uronium or hexafluorophosphonium derivatives, e.g. 1-hydroxy-benzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), ethyl 2-cyano-2-(hydroxyimino)acetate, 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (HATU), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate (H BTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluoroborate (T BTU), 2-succinimido-1,1,3,3-tetramethyluronium-tetrafluoroborate (TSTU), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyl-uronium-tetrafluoroborate (TNTU), O-[(cyano(ethoxycarbonyl)methyliden)amino]-1,1,3,3-tetramethyluronium-tetrafluoroborate (TOTU), O-(benzotriazol-1-yl)-1,3-dimethyl-1,3-dimethylene uronium hexafluorophosphate (HBMDU), O-(benzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), O-(benzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uronium hexafluorophosphate (HBPipU), 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt), 1-hydroxy-7-azabenzotriazole and its corresponding uronium or phosphonium salts, designated HAPyU and AOP, 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), or the like;
Carbodiimides, e.g. dicyclohexylcarbodiimide, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, 1-tert-butyl-3-ethylcarbodiimide, N-cyclohexyl-N′-2-morpholinoethyl)carbodiimide or diisopropylcarbodiimide (especially for ester formation via O-acyl urea formation of the carboxylic group); or
active ester forming agents, e.g. 2-mercaptobenzothiazole (2-MBT),
azide forming agents, e.g. diphenyl phosphoryl azide,
acid anhydrides, such as propane phosphonic acid anhydride,
acid halogenation agents, e.g. 1-chloro-N,N,2-trimethyl-1-propenylamine, chloro-N,N,N′,N′-bis(tetramethylene)formamidinium tetrafluoroborate or hexafluorophosphate, chloro-N,N,N′,N′-tetramethlformamidinium hexafluorophosphate, fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate, fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate,or the like, or mixtures of two or more such agents.
Also for the ester coupling of compounds of the formula XI or XIA with those of the formula XII or XIIA, respectively, the corresponding reactive carboxyl compounds can be used or formed in situ. Here, especially MSNT is preferred as coupling agent as this allows for the maintenance of high stereospecificity.
For the macrolactonization of a compound of the formula III, especially IIIA, also coupling reagents and conditions as described for the coupling of amino acids can be used.
The reactions may, in each case, where appropriate, be conducted in the presence of a mild base (e.g. N-methylmorpholine, a trialkylamine, e.g. ethyldiisopropylamine, a di-(alkyl)aminopyridine, such as N,N-dimethylaminopyridine, or the like (taking care that the conditions are not so basic as to allow for the hydrolysis of ester groups, e.g. the depsipeptide ester group, present in precursors of the compound of the formula I), where appropriate or required in the presence of an appropriate solvent or solvent mixture, e.g. an N,N dialkylformamide, such as dimethylformamide, a halogenated hydrocarbon, e.g. dichloromethane, N-alkylpyrrolidones, such as N-methylpyrrolidone, nitriles, e.g. acetonitrile, ethers, such as dioxane or tetrahydrofurane, or further an aromatic hydrocarbon, e.g. toluene, or mixtures of two or more, where, provided an excess of coupling agent is present, also water may be present. The temperatures may be ambient temperature or lower or higher, e.g. in the range from −20° C. to 50° C.
The amino acids of the formula VII, VIIA, IX, IXA, XI, XIA, XV, XVA, XVI, XVIA, XVII, XVII (obtainable e.g. by Solution Phase peptide synthesis) are known or they can be synthesized according to methods known in the art, they are commercially available, and/or they can be synthesized in analogy to methods known in the art.
Also the remaining starting materials, e.g. the acid of the formula XIV, are known or they can be synthesized according to methods known in the art, they are commercially available, and/or they can be synthesized in analogy to methods known in the art.
Coupling reactions for dipeptides make us of the corresponding carboxylic groups of amino acids in free form or in activated form.